g protein-coupled estrogen receptor-selective ligands modulate
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Hindawi Publishing CorporationObstetrics and Gynecology InternationalVolume 2013, Article ID 472720, 17 pageshttp://dx.doi.org/10.1155/2013/472720
Research ArticleG Protein-Coupled Estrogen Receptor-SelectiveLigands Modulate Endometrial Tumor Growth
Whitney K. Petrie,1 Megan K. Dennis,1 Chelin Hu,1 Donghai Dai,2,3 Jeffrey B. Arterburn,4
Harriet O. Smith,1,2,5 Helen J. Hathaway,1 and Eric R. Prossnitz1
1 Department of Cell Biology and Physiology and UNM Cancer Center, The University of New MexicoHealth Sciences Center, Albuquerque, NM 87131, USA
2Department of Obstetrics and Gynecology, UNM Cancer Center, The University of New MexicoHealth Sciences Center, Albuquerque, NM 87131, USA
3Department of Obstetrics and Gynecology, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA4Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA5Department of Obstetrics and Gynecology and Womens Health, Albert Einstein College of Medicine and MontefioreMedical Center, Bronx, NY 10461, USA
Correspondence should be addressed to Eric R. Prossnitz; email@example.com
Received 6 August 2013; Accepted 17 September 2013
Academic Editor: Andrew P. Bradford
Copyright 2013 Whitney K. Petrie et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Endometrial carcinoma is the most common cancer of the female reproductive tract. GPER/GPR30 is a 7-transmembranespanning G protein-coupled receptor that has been identified as the third estrogen receptor, in addition to ER and ER.High GPER expression is predictive of poor survival in endometrial and ovarian cancer, but despite this, the estrogen-mediatedsignaling pathways and specific estrogen receptors involved in endometrial cancer remain unclear. Here, employing ER-negative Hec50 endometrial cancer cells, we demonstrate that GPER mediates estrogen-stimulated activation of ERK andPI3K via matrix metalloproteinase activation and subsequent transactivation of the EGFR and that ER-targeted therapeuticagents (4-hydroxytamoxifen, ICI182,780/fulvestrant, and Raloxifene), the phytoestrogen genistein, and the ER-selective agonistpropylpyrazole triol also function as GPER agonists. Furthermore, xenograft tumors of Hec50 cells yield enhanced growth withG-1 and estrogen, the latter being inhibited by GPER-selective pharmacologic antagonism with G36. These results have importantimplications with respect to the use of putatively ER-selective ligands and particularly for the widespread long-term use of ER-targeted therapeutics. Moreover, our findings shed light on the potential mechanisms of SERM/SERD side effects reported inmany clinical studies. Finally, our results provide the first demonstration that pharmacological inhibition of GPER activity in vivoprevents estrogen-mediated tumor growth.
Carcinoma of the endometrium is the most common cancerof the female reproductive tract with over 40,000 newdiagnoses and over 7,000 deaths per year in the UnitedStates. Although the majority (75%) of endometrial tumorsare of endometrioid histology (designated type I tumors),expressing high levels of estrogen receptor (ER), progesteronereceptor (PR), and epidermal growth factor receptor (EGFR),
about 25% of tumors are of advanced stage (designatedtype II tumors), are unlikely to be ER+/PR+, and have apoorer prognosis . Although overlap exists with respectto histology, genetic aberations, and epidemiological profiles,the two tumor types appear to represent discrete carcinogenicprocesses with distinct molecular characteristics. Type Itumors consist of well-differentiated tumors preceded byendometrial hyperplasia and are associated with a loss ofPTEN expression as well as abnormalities in -catenin, Kras,
2 Obstetrics and Gynecology International
and DNA mismatch repair genes. Type 2 tumors are a het-erogeneous group of tumors including high-grade (undiffer-entiated) endometrioid carcinomas, uterine papillary serouscarcinomas, clear cell carcinomas, and carcinosarcomas, withdifferent mutational profiles. Over 90% of uterine papillaryserous carcinomas are associated with p53 mutations, 4560% have Her-2/neu mutations, and PTEN mutations arerare [2, 3]. Carcinosarcomas, which are characterized bymalignant epithelial andmesenchymal components, are asso-ciated with many of the epidemiological risk factors linkedto endometrioid carcinomas including obesity and exposureto tamoxifen therapy, suggesting that dysregulated estrogensignaling has a role in their pathogenesis and may representa therapeutic target. Moreover, recent mutational profilingstudies indicate that whereas some carcinosarcomas sharemutations with type 1 tumors (PTEN and ARID1A), othersshare mutations with uterine papillary serous carcinomas(notably p53 and PPP2R1A) .
The lack of estrogen receptor (ER) expression inmost type II tumors has led to the assumption that thesetumors must be estrogen-independent and that treatmentwith antiestrogens (selective estrogen receptor modulators(SERMs) such as tamoxifen and Raloxifene, or pure antag-onists/selective estrogen receptor modulators (SERDs) suchas ICI182,780/fulvestrant) commonly used in breast can-cer treatment, would be ineffectual, a conclusion largelysubstantiated in a number of clinical trials [5, 6]. In fact,prolonged treatment of breast cancer with SERMs such astamoxifen leads to an increased incidence of endometrialcancer , particularly those of high-risk histologic types, resulting in significantly poorer overall survival . Theeffects of tamoxifen in the uterus have been ascribed toaltered expression of nuclear coregulatory proteins in theendometrium compared to the breast, resulting in moderateagonist activity of tamoxifen in the uterus, compared to itsantagonistic effects in the breast . However, recentresults have suggested that a heretofore-underappreciatedestrogen receptor, the G protein-coupled estrogen recep-tor (GPER, formerly GPR30), may play an important rolein both the increased incidence of endometrial cancer inwomen treated with tamoxifen  as well as representingan alternate mechanism through which endometrial cancers,particularly type II tumors, can maintain responsiveness toestrogen .
GPER is a member of the 7-transmembrane spanningG protein-coupled receptor (GPCR) superfamily, struc-turally unrelated to the nuclear receptor family membersER and ER [16, 17]. Activation of GPER by estrogenhas been demonstrated in many cancer cell lines [18, 19],including endometrial cancer cells [15, 2027]. GPER isalso activated by antiestrogens including tamoxifen (i.e., 4-hydroxytamoxifen)  and ICI182,780 (fulvestrant) ,leading to the suggestion that GPER plays a role in hormone-resistance in breast cancer [30, 31] as well as in theincreased incidence of endometrial cancer in women takingtamoxifen for breast cancer [14, 32]. Furthermore, GPER(over)expression has been associated with many cancersand in particular poor prognosis in a number of cancers,including breast , ovarian , lung , pancreatic ,
and endometrial  although observations to the contraryhave also been reported [38, 39].
Because of the lack of specificity of estrogen and anti-estrogens for the three known estrogen receptors (ER, ER,andGPER),we have developed both aGPER-selective agonist(G-1, ) and antagonists (G15  and G36 ) thatdisplay virtually no activity towards the classical estrogenreceptors. In the current study, we examine the expressionand function of GPER in the ER/ER endometrial cancercell line Hec50 [43, 44], which is representative of type IIendometrial cancers. We demonstrate that, in Hec50 cells,GPER is localized predominantly in intracellular membranesand mediates PI3K and ERK activation in response to bothestrogen and G-1 as well as the antiestrogens tamoxifen,Raloxifene, and ICI182,780 and the ER-selective agonistpropylpyrazole triol (PPT). We also demonstrate that Hec50cells and primary patient endometrial adenocarcinomasmaintain expression of GPER when grown as xenografttumors. Finally, we demonstrate both estrogen and G-1stimulate Hec50 xenograft tumor growth in vivo and that theGPER antagonist G36 greatly reduces growth of estrogen-stimulated Hec50 tumors. Overall, these results suggest thatGPER may play a critical role in endometrial carcinogenesis,providing a novel target for prognosis and treatment.
2. Materials and Methods
Reagents. 17-estradiol, 17-estradiol, 4-hydroxytamoxifen,Raloxifene, genistein, LY294002, bovine serum albumin(BSA), normal goat serum, insulin, transferrin, hydrocor-tisone, fetuin, pancreatin, and trypsin were from Sigma(St. Louis, MO, USA). AG1478 and GM6001 were fromCalbiochem (Billerica,MA, USA). DPN, PPT, and ICI182,780were from Tocris Chemicals (Ellisville, MO, USA). G-1,G15, and G36 were synthesized as previously described . Goat anti-rabbit Alexa-488, goat anti-rabbit Alexa-568,and donkey anti-mouse Alexa-568-conjugated secondaryantibodieswere from Invitrogen (Carlsbad, CA,USA). Rabbitanti-GPER C-terminal antiserum (cross-reactive to bothhuman and murine sequences) was produced as previouslydescribed and used at a dilution of 1 : 10,000 . Rab-bit anti-GPER human N-terminal antiserum was producedagainst the peptide sequence MDVTSQARGVGLEMYPG-TAQPAAC (with an added carboxy-terminal cysteine forconjugation to KLH) by New England Peptide, Inc. (Gardner,MA, USA) and used at a dilution of 1 : 5000. Polyclonalantibodies against total ERK and pERK were from Cell Sig-naling (Danvers, MA, USA); monoclonal anti-actin and anti--catenin antibodies were from Millipore (Burlington, MA,USA).